i. Field of the Invention
This invention relates to autoantibodiesassociated with acquired hypoparathyroidism, the autoantigens recognized by these autoantibodies, and to assay methods useful in the detection and treatment of acquired hypoparathyroidism.
ii. Background
Acquired hypoparathyroidism(AH) results from a deficiency of parathyroid hormone (PTH) secretion which leads to hypocalcemia. PTH plays an important role in calcium homeostasis, in that it can enhance calcium resorption from bone, increase calcium reabsorption from the kidney, and act on the renal tubule to promote the conversion of 25-hydroxyvitamin D to its active 1,25-dihydroxy metabolite, which in turn, increases the intestinal calcium absorption (Conklin, 1994; Lund, 1980). The most prominent clinical features of hypocalcemia are muscular irritability, tetany and seizures (Aurbach, 1990).
AH occurs as a common component of type I autoimmune polyglandular syndrome (APS I) which presents in infants or young children. AH also occurs in association with Hashimoto thyroiditis and/or hypothyrodism, a combination which most often affects adult women. APS I is characterized by mucocutaneous candidiasis, hypoparathyroidism and Addison disease, often accompanied by early onset pernicious anemia, chronic active hepatitis, alopecia and primary hypogonadism (Neufeld, 1980; Neufeld, 1981; Winter, 1992, Maclaren, 1985; Muir, 1991). APS I occurs either as an autosomal recessive disease, or as a seemingly sporadic disorder. Whereas APS I is not linked to genes within the HLA-DR region (Ahonen, 1988), the responsible gene has been recently mapped to chromosome 21q22.3 (Aaltonen, 1994). AH may result in mental retardation or unexplained epilepsy if it is not appropriately recognized and treated by the relatively simple and inexpensive expedient of activated vitamin D therapy and calcium supplementation. In APS I, however, normalization of calcium levels through vitamin D supplements may be confounded by malabsorption.
An autoimmune etiology for AH has been suggested because of its association with other autoimmune diseases (Ahonen, 1990), and by reports of autoantibodies directed against the parathyroid tissues in affected individuals. Autoantibodies to the parathyroid glands were first reported by Blizzard et al (Blizzard, 1966). In that study, 38% of 74 patients with autoimmune hypoparathyroidism were found to be positive compared with only 6% of 245 healthy control subjects. The results from subsequent studies were controversial, since the antibodies often appeared to be directed against mitochondrial antigens. These may be false positive results, because mitochondrial rich cells are found within normal parathyroid tissue and anti-mitochondrial antibodies are common in autoimmune disorders (Betterle, 1985). Autoantibodies from the sera of patients with sporadic adult onset hypoparathyroidism however have been reported to bind to the cell surfaces of human parathyroid cells, resulting in an inhibition of PTH secretion (Posillico, 1986). In addition, autoantibodies in the sera of patients with AH have been reported to be cytotoxic for cultured bovine parathyroid cells, by an antibody mediated cytotoxicity dependent on complement fixation and activation (Brandi, 1986; Fattorossi, 1988). Whereas the above findings may suggest the possible involvement of autoantibodies against parathyroid glands in AH, the nature of the targeted autoantigens has only recently been identified by Song et al., Abstract No. OR2-3, 77th Annual Meeting of the Endocrine Society, Washington, D.C., Jun. 14-17, 1995.
The regulation of PTH secretion requires that parathyroid cells sense the serum free ionized calcium levels in extracellular fluid. The recently cloned calcium sensing receptor (CA-SR) responds to increased levels of extracellularcalcium by triggering a phospholipase-C (PLC) dependent pathway which in turn induces the parathyroid cell to decrease its constituent PTH secretion (Conklin, 1994). The CA-SR gene has been mapped to chromosome 3q2 (Pollak, 1993), and the translated protein is a linear peptide of 1085 amino acids, with 7 putative transmembrane-spanningdomains and a large extracellular domain of 613 amino acids at the amino terminus (Brown, 1993). The external domain probably serves as the actual ionized calcium detector. It is a member of a new family of G-protein-coupled receptors which is densely expressed in parathyroid cells, but to some extent also in thyroid C-cells, brain and kidney. Mutations of the CA-SR gene have been found to be responsible for familial benign hypocalciuric hypercalcemia (FBHH) and autosomal dominant hypocalcemia (Pollak, 1993).
The following references are discussed in greater detail below:
1. Betterle, C., et al. (1985) Clin. Exp. Immunol. 62:353-360. PA1 2. Blizzard, R. M., et al. (1966) Clin. Exp. Immunol. 1:119-128. PA1 3. Brandi, E. M., et al. (1986) P.N.A.S. USA 83:8366-8369. PA1 4. Brown, E. M., et al. (1993) Nature 366:575-580. PA1 5. Fattorossi, A., et al. (1988) P.N.A.S. USA 85:4015-4019. PA1 6. Irvine, W. J., et al. (1969) Clin. Exp. Immunol. 4:505-510. PA1 7. Juhlin, C., et al. (1987) P.N.A.S. USA 84:2990-2994.
None of these references teach or suggest that the autoantibodies detected in patients with AH have, as their antigenic target, the calcium sensing receptor expressed by parathyroid chief cells. In addition, none of these references make any mention of the 70 or 80 kilodalton proteins which are part of the instant invention.
The Blizzard reference noted the existence of antibodies in patients with hypoparathyroidism which seemed to react in an organ specific manner with parathyroid tissue. The reactive antigen was not identified, and the authors cast doubt on whether such autoantibodies could have any etiologic relationship to the development of AH. Thus, Blizzard et aL teach away from the instant invention which establishes such a relationship. It is noted, however, that Blizzard et al make reference to publications of Davis, et al. (Lancet ii, 1432, 1961) and of Jones & Fourman (Lancet ii, 119, 1963; Lancet ii, 121, 1963), in support of the suggestion that "a test to detect subclinical hypoparathyroidism . . . would permit detection of partial hypoparathyroidism." The assay method of the instant invention is different from the test taught in those references.
The Irvine reference reported the presence of antibody which reacted with both parathyroid oxyphil and chief cells in a patient with idiopathic hypoparathyroidism. However, the identity of the antigens involved in those immune responses was not determined.
The Betterle reference suggests the existence of anti-mitochondrial autoantibodies in idiopathic hypoparathyroidism,and takes exception to Blizzard et al.'s suggestion that the autoantibodies detected in AH are parathyroid organ specific. The antigen identified by Betterle et al. was a 46 kilodalton protein, clearly different from the antigens identified in the instant invention.
The Brandi et al. reference discloses an antibody in AH patients which was cytotoxic for parathyroid and adrenal cortex cells, but not for pituitary, thyroid, liver or kidney cells. The assay method disclosed depends on cell culture and cytolysis (release of .sup.51 Cr) or immunofluorescence. The antigens involved in the method are not identified. Thus, a specific antigen based assay method is neither disclosed nor suggested.
The Juhlin reference relates to the development of monoclonal antibodies against whole parathyroid cells which exclusively recognize antigens on parathyroid and kidney tubule cells. The authors speculate that the antibodies bind to and block a cell surface calcium sensing receptor on these cells. However, the antibodies used in the study were not autoantibodies, and the nature of the antigen recognized was not defined, other than speculatively, based on the functional changes observed upon antibody binding.
The Fattorossi reference is a follow-up and an extension of the work reported by Brandi et al. The cytotoxic antibody was further classified as an IgM, it was found to not be organ specific, it reacted with endothelial cells, and these researchers found that two target antigens, one of molecular weight 200 kilodaltons and the other of 130 kilodaltons, were recognized. There is no suggestion that the target antigens are related to the parathyroid specific calcium sensing receptor.
The Brown reference discloses the cloning of a bovine parathyroid extracellular calcium sensing receptor having a molecular weight of about 120 kilodaltons. There is no suggestion that there is any relationship between this receptor and autoimmune hypoparathyroidism.
Several patent publications are also discussed below:
1. U.S. Pat. Nos. 5,053,491 and 4,864,020, issued to Cance et al., Washington University:
These patents relate to monoclonal antibodies specific for an antigen present on the surface of parathyroid tissue and are used to identify parathyroid tissue. The antigen is a 191 kilodalton protein (unreduced; reduced molecular weight is about 171 kilodaltons). By contrast, the instant method and compositionsrelate to antigens of 120 kD, 70 kD and 80 kD; and fragments thereof.
2. WO 88/03271 and related foreign equivalents, Juhlin et al.:
This patent publication is related to the above noted Juhlin scientific publication discussed above.
3. WO 94/28019 and related foreign equivalents, Juhlin et al.:
This patent publication relates to the work of Brown et al., discussed above, which disclosed cloning of the bovine calcium sensing receptor. This patent application relates to the cloning of the human calcium sensor, which they refer to as the calcium sensor protein, or CSP. The CSP clone is said to be useful for obtaining agonists/antagoniststo the CSP and for obtaining antibodies to the CSP. The antibodies and agonists/antagonistsso obtained are predicted to be useful in the treatment of a number of disease states including hyperparathyroidism. Thus, like the Brown et al. publication, this publication does not make any connection between the CSP and autoimmune hyparathyroidism. Of interest is the identification of this molecule by the WO 94/28019 applicants as having a molecular weight of 500 kDa. The instant invention, by contrast, relates to the human parathyroid calcium sensing receptor as a 120 kDa molecule.
4. U.S. patent application Ser. No. 07/356,999, U.S. Department of Health and Human Services, Aurbach:
This patent publication relates to the above discussed Brandi et al. and the subsequent Fattorossi et al. publications. This publication refers to a bovine parathyroid cell line in culture which, it is urged, is useful for the characterization of the antibody in autoimmune hypoparathyroidism. A specific antigen based assay method is neither taught nor suggested.
In the paragraph bridging pages 17 and 18 of the reference, it is postulated that the availability of a clone for the 500 kDa protein may assist in studies of autoimmune hypoparathyroidism. A publication of Brown, E. M. (1991) Phys. Rev. 71, 371-411 is cited as implicating autoimmunity in the pathogenesis of rare idiopathic hypoparathyroidism.
By contrast of the above discussed references this invention documents the existence of parathyroid autoantibodies in AH, and characterizes the reacting human specific parathyroidautoantigens. The parathyroid reactive autoantibodies are frequent in AH, and the CA-SR is an important autoantigen target in the disease.
Our finding that parathyroid cell derived CA-SR is a major autoantigen in AH targeted by autoantibodies is a novel observation, adding to the list of autoantigens that are receptors targeted by an immune response in an organ specific autoimmune disease (Wilkin, 1990). The thyroid stimulating hormone (TSH) receptors in autoimmune thyroid disease (Davies, 1981), acetylcholine (ACh) receptors of skeletal muscle in myasthenia gravis (Gonzalez-Ros, 1984; Fumagalli, 1982; and Aesonki, 1981), gastrin receptors in pernicious anemia (Loveridge, 1980), corticotropin receptors in Addison disease (Kendall-Taylor, 1988), and insulin receptors in IDD (Maron, 1983) are important autoantigens to their respective diseases. The mechanisms for involvement of receptors in autoimmune responses are probably complex. The first possibility is that autoantibodies against cell-surface receptors may lead to functional abnormalities of the cells expressing them, resulting in receptor-mediated stimulation or inhibition of the targeted cells. One example of stimulation by an agonist autoantibody mimicking a physiologic molecule is Graves disease which is caused by the binding of autoantibody to TSH receptors such that they are stimulated but in a more prolonged manner than for TSH itself (long acting thyroid stimulator or LATS). One example of inhibition by antagonist autoantibody is myasthenia gravis which is caused by the binding of antibody to ACh receptors. The second possibility is that the autoantibodies cross-link the receptors and increase the rate of their degradation which ultimately lead to their depletion. The third possibility is that the autoantibody can bind to the receptor, fix complement, and thereby induce damage to the cells expressing the receptor. In our study, the autoantibody directed against the external domain of the CA-SR may play a direct role in the pathogenesis of AH as an agonist autoantibody. The autoantibody could induce the same effect as normal serum ionized calcium by binding and activating the receptors which in turn stimulate signal transduction events thereby raising intracellular ionized calcium levels and depressing PTH secretion. That we found the external domain and not the internal domain of the CA-SR to contain the reactive epitope of parathyroid autoantibodies in AH, suggesting a functional role for the autoantibodies. This is also supported by a previous report that autoantibodies from the sera of patients with adult-onset AH bound to the cell surface of human parathyroidcells and induced inhibition of PTH secretion (Posillico, 1986). Alternatively, the autoantibodies could directly induce damage to the PTH-secreting cells. This is consistent with other reports that autoantibodies in the sera of patients with AH were found to be cytotoxic to cultured bovine parathyroid cells by an antibody mediated cytotoxicity (Brandi, 1986; Fattorossi, 1988), and that the epithelial cells of the parathyroid glands in AH patients were either diminished in number or absent by histological studies, sometimes accompanied by a lymphocytic infiltrate. T cells may be contributors to the pathogenesis of AH (Wortsman, 1992), however, we conclude that AH may be predominantly an antibody-mediated disease for which the instant detection and treatment method provides a significant solution.